Structural journey of an insecticidal protein against western corn rootworm

The broad adoption of transgenic crops has revolutionized agriculture. However, resistance to insecticidal proteins by agricultural pests poses a continuous challenge to maintaining crop productivity and new proteins are urgently needed to replace those utilized for existing transgenic traits. We identified an insecticidal membrane attack complex/perforin (MACPF) protein, Mpf2Ba1, with strong activity against the devastating coleopteran pest western corn rootworm (WCR) and a novel site of action. Using an integrative structural biology approach, we determined monomeric, pre-pore and pore structures, revealing changes between structural states at high resolution. We discovered an assembly inhibition mechanism, a molecular switch that activates pre-pore oligomerization upon gut fluid incubation and solved the highest resolution MACPF pore structure to-date. Our findings demonstrate not only the utility of Mpf2Ba1 in the development of biotechnology solutions for protecting maize from WCR to promote food security, but also uncover previously unknown mechanistic principles of bacterial MACPF assembly.


Supplementary Figures
Supplementary Figure 1 | Mpf2Ba1 root expression vs. corn root worm nodal injury score (CRWNIS). a, In planta efficacy data expressed as the corn rootworm node injury score1 as a function of protein accumulation (mass spectrometry) in the root determined using the peptide sequence EPTPPGYTK as described previously in Hu

Supplementary Tables
Supplementary Table 1.
Conservative mutations of Mpf2Ba1-1167 relative to the wild-type protein.  General observations from all locations indicated that the predominant corn rootworm species was the WCR. Combining the data across all four testing locations, plants expressing the Mpf2Ba1 protein provided very good root protection from feeding injury by corn rootworm larvae ( Supplementary Fig. 1b). Details on statistics for the fixed and random effects are summarized in the Material and Methods section. Mean node-injury scores between the 2 Mpf2Ba1 constructs were numerically lower, but not significantly different from the root protection provided by the commercial hybrid corn line DAS-59122-7 across the four locations (P < 0.05; Supplementary Fig.   1b). Both Mpf2Ba1 constructs provided good protection compared with the negative control (P < 0.05, Supplementary Table 1). As reference, a node-injury score of 1.0 under field conditions has been estimated to cause a 15 to 18% reduction in corn grain yield 26,27 .

Activity against resistant WCR
Using an artificial diet, we performed concentration-response bioassays and computed the LC50 values for Mpf2Ba1 against the susceptible rootworm population and the "Readlyn" population (Supplementary Table 3). The Readlyn population is much less sensitive to both Gpp34Ab1/Tpp35Ab1 and mCry3A with resistance ratios (LC50 against Readlyn population/LC50 against susceptible population) of 8-and 43-fold, respectively. Mpf2Ba1 or Mpf2Ba1-1167 shows a resistance ratio of <1-fold, indicating that there is no cross-resistance to mCry3Aa or Gpp34Ab1/Tpp35Ab1. These results show that the mechanism by which Mpf2Ba1 elicits mortality is different compared to either mCry3Aa or Gpp34Ab1/Tpp35Ab1.

Oligomerization of Mpf2Ba1 upon N-terminal cleavage
As stated in the main text, proteolytic activation of Mpf2Ba1 is necessary, but not sufficient to induce oligomerization. Oligomerization was first observed using denaturing polyacrylamide gel electrophoresis after incubation of the protein with gut fluid extracted from WCR larvae ( Supplementary Fig. 5). The oligomerization could be prevented by treating the gut fluid with heat, a protease inhibitor cocktail (cOmplete TM EDTA-free, Roche Lifescience), or with PMSF alone ( Supplementary Fig. 5). However, it was not clear whether the N-terminal truncation per se was triggering oligomerization or not. Therefore, Mpf2Ba1 was treated with trypsin and chymotrypsin to see if these proteases truncated the protein and whether oligomerization was observed. Both proteases truncated Mpf2Ba1 to a fragment that was very similar in size to the one observed with gut fluid processing, but neither caused oligomer formation to occur ( Supplementary Fig. 5).
Trypsin was then used to activate Mpf2Ba1 and the resulting peptide truncated at Ala26 was exposed to gut fluid where oligomerization was observed. Furthermore, gut fluid treated with protease inhibitors could drive oligomer formation of truncated Mpf2Ba1 indicating that additional proteolysis was not needed for oligomerization to occur. These observations indicated that an additional factor must be present in gut fluid that interacts with activated Mpf2Ba1 and triggers oligomerization. Finally, incubation of trypsin-truncated Mpf2Ba1 with BBMVs ( Supplementary   Fig. 5) or with southern corn rootworm cells (Du182A) also resulted in oligomer/pore formation ( Fig. 1b and c), indicating that binding to a membrane receptor triggers oligomerization.

Identification, isolation, and purification of Mpf2Ba1
The insecticidal protein Mpf2Ba1 was identified through activity-based screening and protein placed into each well of a 96 well plate. The assay was run for four days at 25° C and then was scored for insect mortality and stunting of insect growth. The scores were noted as dead "3", severely stunted "2" (little or no growth but alive), stunted "1" (growth to second instar but not equivalent to controls) or normal growth "0". Samples demonstrating mortality or severe stunting were studied further. Once the activity was established, the equivalent sample was subjected to heat and Pronase treatments and resubmitted for insect bioassays to confirm that the insecticidal activity was due to a proteinaceous component.
To identify the bacterial strain where activity was observed, genomic DNA of isolated strain WCR. Note that for preparing plant expression constructs (see section below) the mpf2ba1 gene sequence was modified to include an additional Ala immediately following the translation initiation Met, thus increasing the amino acid reference numbers by one. Except as noted, all of the structure and biochemistry studies described below used this protein sequence.

Generation of Mpf2Ba1-1167
Simulated gastric fluid (SGF) is a common method for assessing the potential allergenicity of nonfood proteins 31 . It was observed that Mpf2Ba1 was stable when exposed to SGF (data not shown). An engineered SGF-susceptible variant, Mpf2Ba1-1167 (accession #OP575916), was generated by making five phenylalanine to tyrosine and four isoleucine to leucine mutations (Supplementary Table 1). Mpf2Ba1-1167 had increased susceptibility to SGF exposure while maintaining wild type potency against WCR (Fig. 1). Mpf2Ba1-1167 was used as a representative of Mpf2Ba1 for crystal structure determination.
Greenhouse efficacy of Mpf2Ba1-expressing plants In construct 2, the transit peptide was:

MLIISSTPHLGVKAWKMVAWPGSARWAKRLSMGPVPVYSAWNQAPMTASMARRPFLISLVR.
The experimental unit in the field was a single-row plot of corn 3 m in length and a row spacing of 76 cm. The experimental design was a randomized complete block with subsamples, planted at 8 locations, with treatments randomized within each of 3 replications. Treatments  Crop Protection, Inc., Greensboro, NC, USA) was applied to seeds in all treatments. This is the labeled rate for control of certain secondary insect pests of corn but does not control CRW.
The source of infested WCR eggs was a non-diapausing colony maintained by the Corteva Insectary Production Research group located in Johnston, IA. Root injury to the treatments was evaluated after the peak period of CRW larval feeding had occurred at each location. Roots were evaluated by digging a sub-sample of 3 roots per plot, washing the root systems clean of soil, and then visually assessing the amount of CRW larval injury (node-injury score) using the Iowa State 0-3 node-injury scale 1 .
A linear mixed model was applied to model node-injury scores across locations. Data for node-injury score (Yijmnks) of location ( ) ! , replication (R)j, construct (P)m, event (E)n, plot (K)k and plant s, were modeled as a function of an overall mean μ, factors for location, location by replication, construct, event, location by construct, location by event, plot within each location ( / ) !" and a residual within each location ( / ) !#$%"& . The model can be specified as: where construct was treated as fixed effect, and all the other effects were treated as independent normally distributed random variables with means of zero. F-tests were used to assess significance for fixed effects. T-tests using standard errors from the model were conducted to compare treatment effects. A difference was considered statistically significant if the P-value of the difference was less than 0.05. All data analysis and comparisons were made in ASReml 3.0 (VSN International, Hemel Hempstead, UK, 2009).

Activity of Mpf2Ba1 against resistant strains of WCR
To demonstrate that Mpf2Ba1 works through a mechanism that is distinct compared to the WCR traits that are currently commercialized, we compared its activity against a laboratory susceptible population of WCR to its activity against a field-collected population from Readlyn, IA, an area where there is high use of commercialized traits (denoted as 'Readlyn'). The Readlyn population has been shown to have signs of resistance to both mCry3Aa and Gpp34Ab1/Tpp35Ab1 (see Supplementary Table 3).

Edman degradation sequencing
N-terminal cleavage was determined through Edman degradation sequencing, as described previously 37 . Untreated Mpf2Ba1 (2 mg) and Mpf2Ba1 processed with WCR gut fluid incubated at 37° C overnight (3 ml of 3 mg/ml Mpf2Ba1 mixture) were separated on a 4-12% Bis-Tris Gel and then transferred to a PVDF membrane using an IBlot® (ThermoFisher) semi-dry transfer system. The membrane was then stained with Sypro TM Ruby (ThermoFisher) to visualize protein bands and bands corresponding to untreated Mpf2Ba1, monomeric Mpf2Ba1, and oligomeric Mpf2Ba1 following gut fluid treatment were cut from the membrane and subjected to sequencing.
Both the monomer and oligomeric bands revealed cleavage of 25 amino acids before Ala26 in Mpf2Ba1.